The laboratory rat is a preferred rodent model in pre-clinical drug studies. Their larger size facilitates procedures otherwise difficult in mice, includng studies using instrumentation, blood sampling, and surgeries, and allows for ten times the amount of tissue collection. Although rats are often more suitable than mice for pharmacological, toxicological, physiological, and many other biological assays, the ease of genetic engineering technologies has made the mouse the preeminent rodent model. However, the recent emergence of new and more precise gene targeting techniques for the rat has resulted in significant growth in the production of genetically-modified rats. In Phase I, we demonstrated the feasibility of combining custom site-specific Xanthomonas TAL Nuclease (XTNTM) [a.k.a. TALEN] technology with spermatogonial stem cells (SSCs) for rapid, cost-effective and precise genome engineering in the rat. Indeed, the Phase I studies enabled Transposagen to launch custom XTNTM and knockout rat production services in 2012, and we have successfully created and delivered custom knockout rats to both industry and academic investigators. In work outside the Phase I project, we paired XTN TALEN technology with piggyBacTM technology to create the Footprint-FreeTM Gene Editing System, the only commercially available system that can engineer as little as a single nucleotide without leaving unwanted mutations and allowing for selection of rare events. For Phase II studies, we propose to use SSC- and Footprint-FreeTM Gene Editing technology to create a suite of rats that would express a specific human CP2D6* allele in the absence of the homologous rat Cyp2d genes (i.e., humanized CYP2D6 rat models). Humans carry a single, but highly polymorphic, p450 CYP2D6 gene. CYP2D6 metabolizes nearly 25% of current drugs and is represented by over 70 variants in the population, which possess a wide range of enzyme activities. As a consequence, adverse drug effects, or lack of drug effect, depend on the specific allele(s) an individual is carrying. Humanized CYP2D6 animal models would be of great value for drug testing. To accomplish this task, we will optimize strategies to simultaneously delete a ~60 kb segment of the rat genome that contains the 5-gene Cyp2d cluster and knock-in a specific human CYP2D6* allele so that it would be placed under transcriptional control of the rat Cyp2d2 promoter to ensure a physiological pattern of expression. Not only would the humanized rat be useful for assessing the consequences of CYP2D6*-specific metabolites in the whole animal, but hepatocytes isolated from these animals would provide a reliable and consistent source of CYP2D6* hepatocytes for in vitro drug metabolism testing. More generally, successful optimization of Footprint-FreeTM Gene Editing protocols for the creation of large-scale humanizing knock-in mutations would enable, for the first time, the possibility of sophisticated and cost-effective genome engineering in many mammalian organisms.